bioRxiv preprint doi: https://doi.org/10.1101/610170; this version posted April 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Identification of new viruses specific to the honey bee mite Varroa destructor Salvador Herrero*, Sandra Coll, Rosa M. González-Martínez, Stefano Parenti, Anabel Millán-Leiva, Joel González-Cabrera* ERI BIOTECMED. Department of Genetics. Universitat de València, Valencia, Spain. *Corresponding authors: Email: [email protected], tel: +34 96 354 3006 Email: [email protected], tel: +34 96 354 3122 Keywords: iflavirus, picornavirus, insobevirus, qPCR, +ssRNA virus 1 bioRxiv preprint doi: https://doi.org/10.1101/610170; this version posted April 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Large-scale colony losses among managed Western honey bees have become a serious threat to the beekeeping industry in the last decade. There are multiple factors contributing to these losses but the impact of Varroa destructor parasitism is by far the most important, along with the contribution of some pathogenic viruses vectored by the mite. So far, more than 20 viruses have been identified infecting the honey bee, most of them RNA viruses. They may be maintained either as covert infections or causing severe symptomatic infections, compromising the viability of the colony. In silico analysis of available transcriptomic data obtained from mites collected in the USA and Europe as well as additional investigation with new samples collected locally allowed the description of three novel RNA viruses. Our results showed that these viruses were widespread among samples and that they were present in the mites and in the bees but with differences in the relative abundance and prevalence. However, we have obtained strong evidence showing that these three viruses were able to replicate in the mite, but not in the bee, suggesting that they are selectively infecting the mite. To our knowledge, this is the first demonstration of Varroa-specific viruses, which open the door to future applications that might help controlling the mite through biological control approaches. 2 bioRxiv preprint doi: https://doi.org/10.1101/610170; this version posted April 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Introduction The ectoparasitic mite Varroa destructor Anderson and Trueman (Arachnida: Acari: Varroidae) is considered a major driver for the seasonal losses of managed Western honey bee (Apis mellifera L.) colonies reported worldwide (Rosenkranz et al. 2010). There are multiple factors contributing to these losses, but the impact of V. destructor is by far the most important in many cases (Roberts et al. 2017). The damage caused by the mite is a result of the combined effect of direct feeding on the fat body of immature and adult bees (Ramsey et al. 2019) and the transmission of an extensive set of viruses that infect and debilitate them (McMenamin and Genersch 2015). This resulted in reduced immune response, low tolerance to pesticides, impaired mobility or flying capacity, reduced weight, morphological deformities, paralysis, etcetera (De Jong et al. 1982; Yang and Cox-Foster 2007; Yang and Cox- Foster 2005). So far, more than 20 viruses have been identified in the honey bees (McMenamin and Genersch 2015), most of them ssRNA viruses. Among them, the Acute bee paralysis virus (ABPV), Deformed wing virus (DWV), Israel acute paralysis virus (IAPV), Kashmir bee virus (KBV), Sacbrood virus (SBV), Slow bee paralysis virus (SBPV), Chronic bee paralysis virus (CBPV), Lake Sinai virus (LSV), Kakugo virus, Varroa destructor virus-1 and Black queen cell virus (BQCV) (Boecking and Genersch 2008; Di Prisco et al. 2011; McMenamin and Genersch 2015; Santillán- Galicia et al. 2014) . However, in absence of V. destructor parasitism, many of these viruses have been found infecting seemingly healthy honey bee colonies, in what are considered as covert infections, that do not cause any detectable clinical impact on them (McMenamin and Genersch 2015). After the host shift of V. destructor from Apis cerana L. to A. mellifera L., the prevalence and pathogenicity of some of the 3 bioRxiv preprint doi: https://doi.org/10.1101/610170; this version posted April 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. aforementioned viruses have increased significantly (Rosenkranz et al. 2010). This combination of viruses’ pathogenicity and the high vectoring capacity of V. destructor is considered for many as one of the main causes of the Colony Collapse Disorder (CCD), responsible for very high losses of colonies in Europe and the USA (Potts et al. 2010; Vanengelsdorp et al. 2009). Thus, it is well documented the ‘relation’ of V. destructor with bee pathogenic viruses but there is very little information available about viruses specific (or pathogenic) for the mite itself. There are no evidences of bee pathogenic viruses causing any impact on V. destructor physiology, even with high loads of some of them recorded in mite virome. There are even viruses discovered in the mite whilst showing no evidence of pathogenicity for it (i.e. VdV-1) (Ongus et al. 2004). Recent research has identified two novel viruses present in V. destructor but not detected in the bees. However, the information available does not show clear evidences of the possible differential specificity of these viruses for V. destructor or the honey bee (Levin et al. 2016). In this paper, we report the identification and phylogenetic characterization of three new viruses in the virome of V. destructor. We show strong evidence indicating that these viruses can replicate in the mite but not in the bee, suggesting that they are selectively infective for the mite without affecting the replication of DWV. Material and Methods Mite samples Mites used in this study were collected directly from the brood of honey bee combs, from hives located nearby Valencia, Spain. Capped cells were opened with a pair of tweezers to extract immature bees and the mites parasitizing them. Bees and mites 4 bioRxiv preprint doi: https://doi.org/10.1101/610170; this version posted April 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. extracted from the same cell were put together in a microcentrifuge tube, snapped frozen in liquid nitrogen and stored at -80 ºC until used for qPCR analysis. Virus discovery and genome sequence analysis Viral sequences were identified in two V. destructor transcriptomes assembled from data downloaded from the SRA (https://www.ncbi.nlm.nih.gov/sra). The mites used to generate these data were collected in the USA (Accession SRS353274) and in the UK (Accession PRJNA531374). A set of invertebrate picornaviral sequences from different families was used as query in Blastx search against the different transcriptomes. Those sequences producing significant hits were manually curated to remove redundant sequences. Sequences with similarity restricted to functional domains like zinc finger domain, helicase domain, and RNA binding domain were removed since they can be in viral as well as in non-viral proteins, and possibly representing non-viral sequences. The genomic structure, gene content, and location of the conserved motifs for the non-structural proteins (helicase, protease and RNA-dependent RNA polymerase), were obtained by comparison with their closest viral species. Phylogenetic analysis RNA-dependent RNA polymerase was selected to establish the phylogenetic relationship of viruses described in this study with the closest viruses and representative members of nearest viral families, retrieved from NCBI GenBank (www.ncbi.nlm.nih.gov/genbank). Protein sequences comprising putative conserved domains for +ssRNA viruses were aligned using MAFFT version 7.409 software employing the E-INS-i algorithm 5 bioRxiv preprint doi: https://doi.org/10.1101/610170; this version posted April 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. (Standley and Katoh 2013). Alignments were examined by eye and subsequently, ambiguously aligned regions were removed using TrimAl program (Capella-Gutiérrez et al. 2009). The most appropriate evolutionary model (best-of-fit model) according with corrected Akaike Information Criterion (AICc) was calculated using Small Modelling Selection software (Longueville et al. 2017). Phylogenetic trees were inferred by maximum likelihood approach implemented in PhyML version 3.1 (Guindon et al. 2010), with the LG substitution model, empirical amino acid frequencies, and a four-category gamma distribution of rate, and using Subtree Pruning and Regrafting (SPR) branch-swapping. Branch support was assessed using the approximate likelihood ratio test (aLRT) with the Shimodaira–Hasegawa- like procedure as implemented in PhyML. Branches with <0.6 aLRT support were collapsed using TreeGraph software (Stöver and Müller 2010). Virus detection and quantification Presence